Heterotrimeric G proteins, consisting of alpha, beta and gamma subunits, couple ligand-bound seven transmembrane domain receptors (GPCRs or G-protein coupled receptors) to the regulation of effector proteins and production of intracellular second messengers such as cAMP, cGMP, and Ca2+. G protein signaling mediates the perception of environmental cues in all higher eukaryotic organisms, including yeast, Dictyostelium, plants, and animals. Agonist-bound sensory receptors catalyze the exchange of GTP for GDP on the surface of the Gα subunit to initiate intracellular responses to extracellular signals. Intracellular signaling is mediated through various effector enzymes, including cGMP phosphodiesterase, phospholipase C, adenylate cyclase, etc. (see Kinnamon & Margolskee, 1996, Curr. Opinion Neurobiol. 6: 506–513). Most effector proteins interact with the Gα, although Gβγ subunits also contribute to the specificity of receptor-G protein coupling (Xu et al., 1998, J. Biol. Chem. 273(42): 27275–79).
The G protein α subunits are grouped into four families, Gαs, Gαi, Gαq, and Gα12 according to their sequence homologies and functional similarities. The Gαq family members couple a large group of GPCRs to phospholipase C. Activation of Gαq coupled GPCRs induces intracellular calcium release and the capacitative calcium entry from extracellular space. The consequential increase of cytosolic calcium concentration can be effectively detected by using synthetic or genetically-engineered fluorescent calcium indicators, bioluminescent calcium indicators, calcium-activated ion currents, and by monitoring calcium-regulated gene transcription. Assays based on such calcium readout are available in high-throughput screening (HTS) format.
Signaling specificity among α subunits of the same class having similar biochemical functions is not well understood in vivo. For instance, the Gαq (Gq) class includes four proteins expressed in mammals, called Gαq, Gα11, Gα14, and Gα15 (in mice, Gα16 in humans). Whereas orthologs of these subunits are highly conserved across species (99, 97, 96 and 85% identity, respectively), paralogs of these subunits (expressed in the same species) are not as conserved. This suggests that each type of subunit in the Gq class has a distinct function, however, when transfected into Sf9 cells, the subunits stimulated phospholipase C with similar potency and showed similar activities (Nakamura et al., 1995, J. Biol. Chem. 270: 6246–6253). Xu and colleagues subsequently showed by gene knockouts in mice that Gq60 subunits promiscuously couple to several different receptors in various cell types (1998, J. Biol. Chem. 273(42): 27275).
The promiscuity of the Gαq subclass of G protein subunits provides a valuable tool for analyzing the role of G protein complexes and GPCRs in chemosensory transduction. For instance, the ability of Gαq proteins to bypass the selectivity of the receptor G-protein interaction can be used to study the molecular mechanism of receptor-induced G-protein activation. In addition, the promiscuity toward receptors may be helpful in identifying ligands corresponding to orphan receptors whose signaling properties are unknown. Promiscuous G protein subunits play a particularly useful role in generating screening assays for high affinity GPCR agonists, antagonists, and modulators of chemosensory activity, in that using a single G protein coupler removes the variability of the G protein from the equation, thereby simplifying interpretation of results gleaned from various modulating compounds and GPCRs. Chemosensory modulating compounds involved in taste and/or smell, for instance, could then be used in the pharmaceutical and food industries to customize taste or aroma. In addition, such chemosensory molecules could be used to generate topographic maps that elucidate the relationship between the taste cells of the tongue or olfactory receptors (Ors) and sensory neurons leading to the brain.
Despite their promiscuity, however, Gαq class subunits do not mediate all GPCR—effector interactions. For instance, human Gα16 and its murine counterpart Gα15 are promiscuous G proteins in that they couple to GPCRs of different G protein families (Offermanns and Simon, 1995; Negulescu et al., 1997). However, they are not true universal adapters for GPCRs in that there are at least 11 GPCRs reported to be incapable of activating Gα15/Gα16 (Wu et al., 1992; Arai et al., 1996; Kuang et al., 1996; Lee et al., 1998; Parmentier et al., 1998; Mody et al., 2000). Similar problems arise when using Gα15/α16 to identify ligands of ORs and T2Rs (bitter taste receptors) in that (1) calcium responses to odorants are small and quickly desensitized for ORs in Gα15/α16 transiently transfected cells (Krautwurst et al., 1998); (2) most T2Rs remain orphan using cell lines stably transfected with Gα15 (Adler et al., 2000; Chandrashekar et al., 2000); and (3) threshold concentration of denatonium measured is at least one order higher than expected for bitter receptors, hT2R4 and mT2R8 expressed in cells stably transfected with Gα15 (Adler et al., 2000; Chandrashekar et al., 2000). These problems suggest that the coupling efficiency between ORs/T2Rs and Gα15/α16 is weak and may vary within the family of ORs and T2Rs.
Given the partial promiscuity of Gαq class proteins, it would be desirable to identify or create Gα protein subunits that are more promiscuous than their native counterparts, and which are capable of interacting with a wider variety GPCRs.